Mass spectrometric determination of eicosapentaenoic acid and docosahexaenoic acid

ABSTRACT

The invention relates to the detection of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). In a particular aspect, the invention relates to methods for detecting DHA and EPA by mass spectrometry and kits for carrying out such methods.

CROSS-REFERENCED TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/635,509 filed Jun. 28, 2017, now U.S. Pat. No. 10,534,008, which is acontinuation of U.S. application Ser. No. 14/816,586 filed Aug. 3, 2015,now U.S. Pat. No. 9,696,325, which is a continuation of U.S. applicationSer. No. 14/464,336 filed Aug. 20, 2014, now U.S. Pat. No. 9,097,732,which is a continuation of U.S. application Ser. No. 14/025,503 filedSep. 12, 2013, now abandoned, which is a continuation of U.S.application Ser. No. 13/233,773 filed Sep. 15, 2011, now U.S. Pat. No.8,557,593, which claims the benefit of priority under 35 U.S.C. § 119(e)to U.S. Provisional Application Ser. No. 61/383,695 filed Sep. 16, 2010,each of which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The invention relates to the quantitative measurement of docosahexaenoicacid (DHA) and eicosapentaenoic acid (EPA). In a particular aspect, theinvention relates to methods for quantitative measurement of DHA and EPAby APCI-mass spectrometry.

BACKGROUND OF THE INVENTION

Omega-3 fatty acids are a family of unsaturated fatty acids with acarbon-carbon double bond at the third bond from the methyl end of thefatty acid. The human body cannot synthesize omega-3 fatty acids denovo. Instead, they are obtained in the human diet from certain fish,such as cod, mackerel, herring, salmon, and sardines. Nutritionallyimportant omega-3 fatty acids include docosahexaenoic acid (DHA) andeicosapentaenoic acid (EPA). EPA acts as a precursor for prostaglandin-3(which inhibits platelet aggregation), thromboxane-3, and leukotriene-5.DHA is metabolized to form the docosanoids, which comprise severalfamilies of hormones. DHA is also a major fatty acid in sperm and brainphospholipids. High levels of both EPA and DHA have been linked toreduced triglycerides, heart rate, blood pressure, and atherosclerosis.Decreased levels of EPA have been linked to depression andschizophrenia. Decreased levels of DHA have been linked to Alzheimer'sdisease.

EPA, also sometimes known as timnodonic acid or by the shorthand name20:5(n-3), is a carboxylic acid with a 20-carbon chain with 5-cis doublebonds, the first of which is located at the third carbon from the omegaend of the carbon chain. EPA has a molecular mass of approximately302.451 g/mol.

DHA, also sometimes known as cervonic acid or by the shorthand name22:6(n-3), is a carboxylic acid with a 22-carbon chain with 6-cis doublebonds, the first of which is located at the third carbon from the omegaend of the carbon chain. DHA has a molecular mass of approximately328.488 g/mol.

Quantitation of EPA and DHA by liquid chromatography-mass spectrometrywith ESI has been reported. For example Salm, et al., BiomedChromatogr., 2010, Epub ahead of print, report quantitation of EPA andDHA in plasma by HPLC-ESI (negative ion)-MS/MS; Lacaze, et al., J.Chromatog. A, 2007, 1145:51-57 report methods for quantitating freefatty acids in shellfish tissue extracts with an LC-ESI (negativeion)-MS method; and Zehethofer, et al., Rapid Communications in MassSpectrom., 2008, 22:2125-33 report quantitating free fatty acids inplasma with an UPLC-ESI (positive ion)-MS/MS method. Other massspectrometric methods for quantitation of EPA and/or DHA derivatives,metabolites, or oxidation products have also been reported. For example,Řezanka, reports quantitation of EPA and DHA by preparation of methylesters of EPA and DHA followed by detection of the methyl esters byHPLC-APCI (negative ion)-MS. (See Řezanka, Tomáš, J. High Resol.Chromatogr. 2000, 23:338-42 (EPA and DHA in linseed oil and preparedstandards); and Řezanka, Tomáš, Biochemical Systematics and Ecology2000, 28:847-56 (EpA and DHA in three freshwater crustacean species.)Additionally, Fer, et al., J. Lipid Research 2008, 49:2379-89, reportsdetection of ω- and (ω-1)-hydroxylated derivatives of EPA and DHA by anHPLC-APCI (negative ion)-MS method; Yin, et al., J. Biol. Chem. 2007,282:29890-901, report detection of autoxidation products of EPA and DHAby an HPLC-APCI (negative ion)-MS method; Lawson, et al., J. LipidResearch 2006, 47:2515-24, and Gao, et al., J. Biol. Chem. 2007,282:2529-37, report detection of oxidized derivatives of EPA and DHAwith LC-ESI (negative ion)-MS/MS.

SUMMARY OF THE INVENTION

The present invention provides methods for detecting the amount of DHAand/or EPA in a sample by mass spectrometry.

In one aspect, the invention provides methods for determining the amountof DHA in a sample by mass spectrometry. In some embodiments, themethods include the steps of: (i) subjecting DHA in the sample to anatmospheric pressure chemical ionization (APCI) source to generate oneor more DHA ions detectable by mass spectrometry; (ii) determining theamount of one or more DHA ions by mass spectrometry; and (iii) relatingthe amount of one or more DHA ions to the amount of DHA in the sample.In some embodiments, one or more DHA ions comprise an ion with a mass tocharge ratio (m/z) of about 327.2±0.5. In some embodiments, the samplecomprises a sample derived from a human, such as a human body fluid,such as plasma or serum. In some embodiments, the method has a limit ofdetection for DHA in human serum of about 5 μmol/L or less. In someembodiments, the method further comprises simultaneously determining theamount of EPA in the sample by: (i) subjecting eicosapentaenoic acid(EPA) in the sample to the APCI source to generate one or more EPA ionsdetectable by mass spectrometry; (ii) determining the amount of one ormore EPA ions by mass spectrometry; and (iii) relating the amount of oneor more EPA ions to the amount of a EPA in the sample.

In a second aspect, the invention provides methods for determining theamount of EPA in a sample by mass spectrometry. In some embodiments, themethods include the steps of: (i) subjecting EPA in the sample to anAPCI source to generate one or more EPA ions detectable by massspectrometry; (ii) determining the amount of one or more EPA ions bymass spectrometry; and (iii) relating the amount of one or more EPA ionsto the amount of EPA in the sample. In some embodiments, one or more EPAions comprise an ion with a mass to charge ratio (m/z) of about301.2±0.5. In some embodiments, the sample comprises a sample derivedfrom a human, such as a human body fluid, such as plasma or serum. Insome embodiments, the method has a limit of detection for EPA in humanserum of about 10 μmol/L or less. In some embodiments, the methodfurther comprises simultaneously determining the amount of EPA in saidsample by: (i) subjecting eicosapentaenoic acid (EPA) in the sample tothe APCI source to generate one or more EPA ions detectable by massspectrometry; (ii) determining the amount of said one or more EPA ionsby mass spectrometry; and (iii) relating the amount of one or more EPAions to the amount of a DHA in the sample.

In some embodiments of either of the above two aspects, the APCIionization source is operated in negative ionization mode. In someembodiments, DHA and/or EPA in a sample are subjected to a hydrolyzingagent, such as an acid, prior to ionization. In some embodiments, DHAand/or EPA in a sample are subjected to liquid/liquid extraction priorto ionization. In some embodiments, DHA and/or EPA in a sample aresubjected to liquid chromatography column, such as a high performanceliquid chromatography column, prior to ionization.

In a third aspect, the invention provides methods for determining theamount of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), orboth in a plasma or serum sample from a human patient, comprising: (i)subjecting a serum sample to a hydrolyzing agent to generate ahydrolyzed sample; (ii) purifying DHA and/or EPA in the hydrolyzedsample; (iii) subjecting the purified DHA and/or EPA to an ionizationsource in negative ion mode to generate one or more DHA and/or EPA ionsdetectable by mass spectrometry; (iv) determining the amount of one ormore DHA and/or EPA ions by mass spectrometry; and (v) relating theamount of DHA and/or EPA ions to the amount of a DHA and/or EPA in thesample. In some embodiments, the step of purifying comprises subjectingDHA and/or EPA in said hydrolyzed sample to liquid/liquid extraction. Insome embodiments, the step of purifying comprises subjecting DHA and/orEPA in said hydrolyzed sample to liquid chromatography. In someembodiments, the ionizaoin source is APCI. In some embodiments, one ormore DHA ions comprise an ion with a mass to charge ratio of 327.2±0.5.In some embodiments, one or more EPA ions comprise an ion with a mass tocharge ratio of 301.2±0.5. In some embodiments, DHA and EPA aresimultaneously determined in the human serum sample.

In the some embodiments of the methods described herein, massspectrometry is not conducted by tandem mass spectrometry. In thesemethods, mass spectrometry may be single mass spectrometry conducted byany method known in the art including single ion monitoring orcollecting all data over a range of mass to charge ratios (i.e.,scanning). In some embodiments, mass spectrometry may be conducted bytandem mass spectrometry. In embodiments utilizing tandem massspectrometry, tandem mass spectrometry may be conducted by any methodknown in the art, including for example, multiple reaction monitoring,precursor ion scanning, or product ion scanning.

In embodiments which utilize two or more of an extraction column, ananalytical column, and an ionization source, two or more of thesecomponents may be connected in an on-line fashion to allow for automatedsample processing and analysis.

In embodiments where a sample comprises both EPA and DHA, both analytesin a sample may be ionized and/or detected simultaneously. As usedherein, the term “simultaneous” as applied to simultaneously ionizingand/or detecting the amount of two or more analytes from a sample meansionizing two or more analytes and/or acquiring data reflective of theamount of the two or more analytes in the sample from the same sampleinjection. The data for each analyte may be acquired sequentially or inparallel, depending on the instrumental techniques employed. Forexample, a single sample containing two analytes may be injected into anon-line HPLC column, which may then elute each analyte one after theother, resulting in introduction of the analytes into a massspectrometer sequentially. Determining the amount of each of these twoanalytes is simultaneous for the purposes herein, as both analytesresult from the same sample injection into the HPLC.

In some embodiments, EPA ions detected by mass spectrometry compriseions with a mass/charge ratio (m/z) of 301.2±0.5, and DHA ions detectedby mass spectrometry comprise ions with a mass/charge ratio (m/z) of327.2±0.5.

EPA and DHA may be found in the circulation of an animal and/or may begenerated by a biological organism, such as a plant, an animal, orsingle-celled organism. As such, preferred samples may be biologicalsamples; particularly biological fluid samples such as serum or plasma.In some embodiments, the biological samples may be derived from a humanpatient having or at risk to develop any of the following conditions:pregnancy, cardiac disease, cancer, Alzheimer's, infant cognitivedevelopment deficiencies, infant visual development deficiencies,postpartum depression, dementia, and hypertension.

As used herein, “derivatizing” means reacting two or more molecules toform a new molecule. As used herein, the names of derivatized forms ofcompounds (including fatty acids such as EPA and DHA) include anindication as to the nature of derivatization. For example, the methylesters of EPA and DHA would be referred to as EPA-methyl ester andDHA-methyl ester.

Mass spectrometry may be performed in negative ion mode. Alternatively,mass spectrometry may be performed in positive ion mode. Variousionization sources, including for example atmospheric pressure chemicalionization (APCI), laser diode thermal desorption (LDTD), orelectrospray ionization (ESI), may be used in embodiments of the presentinvention. In certain preferred embodiments, EPA and DHA are measuredusing APCI in negative ion mode.

In preferred embodiments, one or more separately detectable internalstandards are provided in the sample, the amount of which are alsodetermined in the sample. In these embodiments, all or a portion of boththe analyte(s) of interest and the internal standard(s) present in thesample are ionized to produce a plurality of ions detectable in a massspectrometer, and one or more ions produced from each analyte ofinterest and internal standard are detected by mass spectrometry.Exemplary internal standards for EPA and DHA include EPA-²H₅ andDHA-²H_(5,) respectively.

Ions detectable in a mass spectrometer may be generated for each of theexemplary internal standards listed above. Exemplary spectra generateddemonstrating detection of EPA-²H₅ and DHA-²H₅ are discussed in Example4, and shown in FIGS. 3 and 4, respectively.

As used herein, an “isotopic label” produces a mass shift in the labeledmolecule relative to the unlabeled molecule when analyzed by massspectrometric techniques. Examples of suitable labels include deuterium(d or ²H), ¹³C, and ¹⁵N. For example, EPA-²H₅ and DHA-²H₅ have masses ofabout 5 mass units higher than EPA and DHA. The isotopic label can beincorporated at one or more positions in the molecule and one or morekinds of isotopic labels can be used on the same isotopically labeledmolecule.

In other embodiments, the amount of DHA and/or EPA ions may bedetermined by comparison to one or more external reference standards.Exemplary external reference standards include blank plasma or serumspiked with one or more of DHA-²H₅ and EPA-²H₅. External standardstypically will undergo the same treatment and analysis as any othersample to be analyzed.

In certain embodiments, the lower limit of quantitation (LLOQ) of DHA isless than 10 μmol/L; such as between about 10 μmol/L and 0.91 μmol/L;such as between about 5 μmol/L and 0.91 μmol/L; such as between about 2μmol/L and 0.91 μmol/L; such as about 0.91 μmol/L. In certainembodiments, the lower limit of quantitation (LLOQ) of EPA is less than20 μmol/L; such as between about 20 μmol/L and 2.28 μmol/L; such asbetween about 10 μmol/L and 2.28 μmol/L; such as between about 5 μmol/Land 2.28 μmol/L; such as about 2.28 μmol/L.

In certain embodiments, the limit of detection (LOD) of DHA is less than5 μmol/L; such as between about 5 μmol/L and 0.25 μmol/L; such asbetween about 2 μmol/L and 0.25 μmol/L; such as between about 1 μmol/Land 0.25 μmol/L; such as between about 0.5 μmol/L and 0.25 μmol/L; suchas about 0.25 μmol/L. In certain embodiments, the limit of detection(LOD) of EPA is less than 10 μmol/L; such as between about 10 μmol/L and0.67 μmol/L; such as between about 5 μmol/L and 0.67 μmol/L; such asbetween about 2 μmol/L and 0.67 μmol/L; such as between about 1 μmol/Land 0.67 μmol/L; such as about 0.67 μmol/L.

As used herein, unless otherwise stated, the singular forms “a,” “an,”and “the” include plural reference. Thus, for example, a reference to “aprotein” includes a plurality of protein molecules.

As used herein, the term “purification” or “purifying” does not refer toremoving all materials from the sample other than the analyte(s) ofinterest. Instead, purification refers to a procedure that enriches theamount of one or more analytes of interest relative to other componentsin the sample that may interfere with detection of the analyte ofinterest. Purification of the sample by various means may allow relativereduction of one or more interfering substances, e.g., one or moresubstances that may or may not interfere with the detection of selectedparent or daughter ions by mass spectrometry. Relative reduction as thisterm is used does not require that any substance, present with theanalyte of interest in the material to be purified, is entirely removedby purification.

As used herein, the term “solid phase extraction” or “SPE” refers to aprocess in which a chemical mixture is separated into components as aresult of the affinity of components dissolved or suspended in asolution (i.e., mobile phase) for a solid through or around which thesolution is passed (i.e., solid phase). In some instances, as the mobilephase passes through or around the solid phase, undesired components ofthe mobile phase may be retained by the solid phase resulting in apurification of the analyte in the mobile phase. In other instances, theanalyte may be retained by the solid phase, allowing undesiredcomponents of the mobile phase to pass through or around the solidphase. In these instances, a second mobile phase is then used to elutethe retained analyte off of the solid phase for further processing oranalysis. SPE, including TFLC, may operate via a unitary or mixed modemechanism. Mixed mode mechanisms utilize ion exchange and hydrophobicretention in the same column; for example, the solid phase of amixed-mode SPE column may exhibit strong anion exchange and hydrophobicretention; or may exhibit column exhibit strong cation exchange andhydrophobic retention.

As used herein, the term “chromatography” refers to a process in which achemical mixture carried by a liquid or gas is separated into componentsas a result of differential distribution of the chemical entities asthey flow around or over a stationary liquid or solid phase.

As used herein, the term “liquid chromatography” or “LC” means a processof selective retardation of one or more components of a fluid solutionas the fluid uniformly percolates through a column of a finely dividedsubstance, or through capillary passageways. The retardation resultsfrom the distribution of the components of the mixture between one ormore stationary phases and the bulk fluid, (i.e., mobile phase), as thisfluid moves relative to the stationary phase(s). Examples of “liquidchromatography” include reverse phase liquid chromatography (RPLC), highperformance liquid chromatography (HPLC), and turbulent flow liquidchromatography (TFLC) (sometimes known as high turbulence liquidchromatography (HTLC) or high throughput liquid chromatography).

As used herein, the term “high performance liquid chromatography” or“HPLC” (sometimes known as “high pressure liquid chromatography”) refersto liquid chromatography in which the degree of separation is increasedby forcing the mobile phase under pressure through a stationary phase,typically a densely packed column.

As used herein, the term “turbulent flow liquid chromatography” or“TFLC” (sometimes known as high turbulence liquid chromatography or highthroughput liquid chromatography) refers to a form of chromatographythat utilizes turbulent flow of the material being assayed through thecolumn packing as the basis for performing the separation. TFLC has beenapplied in the preparation of samples containing two unnamed drugs priorto analysis by mass spectrometry. See, e.g., Zimmer et al., J ChromatogrA 854: 23-35 (1999); see also, U.S. Pat. Nos. 5,968,367, 5,919,368,5,795,469, and 5,772,874, which further explain TFLC. Persons ofordinary skill in the art understand “turbulent flow”. When fluid flowsslowly and smoothly, the flow is called “laminar flow”. For example,fluid moving through an HPLC column at low flow rates is laminar. Inlaminar flow the motion of the particles of fluid is orderly withparticles moving generally in straight lines. At faster velocities, theinertia of the water overcomes fluid frictional forces and turbulentflow results. Fluid not in contact with the irregular boundary “outruns”that which is slowed by friction or deflected by an uneven surface. Whena fluid is flowing turbulently, it flows in eddies and whirls (orvortices), with more “drag” than when the flow is laminar. Manyreferences are available for assisting in determining when fluid flow islaminar or turbulent (e.g., Turbulent Flow Analysis: Measurement andPrediction, P. S. Bernard & J. M. Wallace, John Wiley & Sons, Inc.,(2000); An Introduction to Turbulent Flow, Jean Mathieu & Julian Scott,Cambridge University Press (2001)).

As used herein, the term “gas chromatography” or “GC” refers tochromatography in which the sample mixture is vaporized and injectedinto a stream of carrier gas (as nitrogen or helium) moving through acolumn containing a stationary phase composed of a liquid or aparticulate solid and is separated into its component compoundsaccording to the affinity of the compounds for the stationary phase.

As used herein, the term “large particle column” or “extraction column”refers to a chromatography column containing an average particlediameter greater than about 50 μm.

As used herein, the term “analytical column” refers to a chromatographycolumn having sufficient chromatographic plates to effect a separationof materials in a sample that elute from the column sufficient to allowa determination of the presence or amount of an analyte. In a preferredembodiment the analytical column contains particles of about 5 μm indiameter. Such columns are often distinguished from “extractioncolumns”, which have the general purpose of separating or extractingretained material from non-retained materials in order to obtain apurified sample for further analysis.

As used herein, the terms “on-line” and “inline”, for example as used in“on-line automated fashion” or “on-line extraction” refers to aprocedure performed without the need for operator intervention. Incontrast, the term “off-line” as used herein refers to a procedurerequiring manual intervention of an operator. Thus, if samples aresubjected to precipitation, and the supernatants are then manuallyloaded into an autosampler, the precipitation and loading steps areoff-line from the subsequent steps. In various embodiments of themethods, one or more steps may be performed in an on-line automatedfashion.

As used herein, the term “mass spectrometry” or “MS” refers to ananalytical technique to identify compounds by their mass. MS refers tomethods of filtering, detecting, and measuring ions based on theirmass-to-charge ratio, or “m/z”. MS technology generally includes (1)ionizing the compounds to form charged species; and (2) detecting thecharged species based on their mass-to-charge ratio. The compounds maybe ionized and detected by any suitable means. A “mass spectrometer”generally includes an ionizer and an ion detector. In general, one ormore molecules of interest are ionized, and the ions are subsequentlyintroduced into a mass spectrometric instrument where, due to acombination of magnetic and electric fields, the ions follow a path inspace that is dependent upon mass (“m”) and charge (“z”). See, e.g.,U.S. Pat. No. 6,204,500, entitled “Mass Spectrometry From Surfaces;”U.S. Pat. No. 6,107,623, entitled “Methods and Apparatus for Tandem MassSpectrometry;” U.S. Pat. No. 6,268,144, entitled “DNA Diagnostics BasedOn Mass Spectrometry;” U.S. Pat. No. 6,124,137, entitled“Surface-Enhanced Photolabile Attachment And Release For Desorption AndDetection Of Analytes;” Wright et al., Prostate Cancer and ProstaticDiseases 1999, 2: 264-76; and Merchant and Weinberger, Electrophoresis2000, 21: 1164-67.

As used herein, the term “operating in negative ion mode” refers tothose mass spectrometry methods where negative ions are generated anddetected. The term “operating in positive ion mode” as used herein,refers to those mass spectrometry methods where positive ions aregenerated and detected.

As used herein, the term “ionization” or “ionizing” refers to theprocess of generating an analyte ion having a net electrical chargeequal to one or more electron units. Negative ions are those having anet negative charge of one or more electron units, while positive ionsare those having a net positive charge of one or more electron units.

As used herein, the term “electron ionization” or “EI” refers to methodsin which an analyte of interest in a gaseous or vapor phase interactswith a flow of electrons. Impact of the electrons with the analyteproduces analyte ions, which may then be subjected to a massspectrometry technique.

As used herein, the term “chemical ionization” or “CI” refers to methodsin which a reagent gas (e.g. ammonia) is subjected to electron impact,and analyte ions are formed by the interaction of reagent gas ions andanalyte molecules.

As used herein, the term “fast atom bombardment” or “FAB” refers tomethods in which a beam of high energy atoms (often Xe or Ar) impacts anon-volatile sample, desorbing and ionizing molecules contained in thesample. Test samples are dissolved in a viscous liquid matrix such asglycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crown ether,2-nitrophenyloctyl ether, sulfolane, diethanolamine, andtriethanolamine. The choice of an appropriate matrix for a compound orsample is an empirical process.

As used herein, the term “matrix-assisted laser desorption ionization”or “MALDI” refers to methods in which a non-volatile sample is exposedto laser irradiation, which desorbs and ionizes analytes in the sampleby various ionization pathways, including photo-ionization, protonation,deprotonation, and cluster decay. For MALDI, the sample is mixed with anenergy-absorbing matrix, which facilitates desorption of analytemolecules.

As used herein, the term “surface enhanced laser desorption ionization”or “SELDI” refers to another method in which a non-volatile sample isexposed to laser irradiation, which desorbs and ionizes analytes in thesample by various ionization pathways, including photo-ionization,protonation, deprotonation, and cluster decay. For SELDI, the sample istypically bound to a surface that preferentially retains one or moreanalytes of interest. As in MALDI, this process may also employ anenergy-absorbing material to facilitate ionization.

As used herein, the term “electrospray ionization” or “ESI,” refers tomethods in which a solution is passed along a short length of capillarytube, to the end of which is applied a high positive or negativeelectric potential. Solution reaching the end of the tube is vaporized(nebulized) into a jet or spray of very small droplets of solution insolvent vapor. This mist of droplets flows through an evaporationchamber, which is heated slightly to prevent condensation and toevaporate solvent. As the droplets get smaller the electrical surfacecharge density increases until such time that the natural repulsionbetween like charges causes ions as well as neutral molecules to bereleased.

As used herein, the term “atmospheric pressure chemical ionization” or“APCI,” refers to mass spectrometry methods that are similar to ESI;however, APCI produces ions by ion-molecule reactions that occur withina plasma at atmospheric pressure. The plasma is maintained by anelectric discharge between the spray capillary and a counter electrode.Then ions are typically extracted into the mass analyzer by use of a setof differentially pumped skimmer stages. A counterflow of dry andpreheated N₂ gas may be used to improve removal of solvent. Thegas-phase ionization in APCI can be more effective than ESI foranalyzing less-polar species.

The term “atmospheric pressure photoionization” or “APPI” as used hereinrefers to the form of mass spectrometry where the mechanism for thephotoionization of molecule M is photon absorption and electron ejectionto form the molecular ion M+. Because the photon energy typically isjust above the ionization potential, the molecular ion is lesssusceptible to dissociation. In many cases it may be possible to analyzesamples without the need for chromatography, thus saving significanttime and expense. In the presence of water vapor or protic solvents, themolecular ion can extract H to form MH+. This tends to occur if M has ahigh proton affinity. This does not affect quantitation accuracy becausethe sum of M+ and MH+ is constant. Drug compounds in protic solvents areusually observed as MH+, whereas nonpolar compounds such as naphthaleneor testosterone usually form M+. See, e.g., Robb et al., Anal. Chem.2000, 72(15): 3653-3659.

As used herein, the term “inductively coupled plasma” or “ICP” refers tomethods in which a sample interacts with a partially ionized gas at asufficiently high temperature such that most elements are atomized andionized.

As used herein, the term “desorption” refers to the removal of ananalyte from a surface and/or the entry of an analyte into a gaseousphase. Laser diode thermal desorption (LDTD) is a technique wherein asample containing the analyte is thermally desorbed into the gas phaseby a laser pulse. The laser hits the back of a specially made 96-wellplate with a metal base. The laser pulse heats the base and the heatcauses the sample to transfer into the gas phase. The gas phase samplemay then be drawn into an ionization source, where the gas phase sampleis ionized in preparation for analysis in the mass spectrometer. Whenusing LDTD, ionization of the gas phase sample may be accomplished byany suitable technique known in the art, such as by ionization with acorona discharge (for example by APCI).

As used herein, the term “field desorption” refers to methods in which anon-volatile test sample is placed on an ionization surface, and anintense electric field is used to generate analyte ions.

As used herein, the term “selective ion monitoring” is a detection modefor a mass spectrometric instrument in which only ions within arelatively narrow mass range, typically about one mass unit, aredetected.

As used herein, “multiple reaction mode,” sometimes known as “selectedreaction monitoring,” is a detection mode for a mass spectrometricinstrument in which a precursor ion and one or more fragment ions areselectively detected.

As used herein, the term “lower limit of quantification”, “lower limitof quantitation” or “LLOQ” refers to the point where measurements becomequantitatively meaningful. The analyte response at this LLOQ isidentifiable, discrete and reproducible with a concentration at whichthe standard deviation (SD) is less than one third of the totalallowable error (TEa; arbitrarily set for DHA and EPA as 22% of theLLOQ).

As used herein, the term “limit of detection” or “LOD” is the point atwhich the measured value is larger than the uncertainty associated withit. The LOD is the point at which a value is beyond the uncertaintyassociated with its measurement and is defined as the mean of the blankplus four times the standard deviation of the blank.

As used herein, an “amount” of an analyte in a body fluid sample refersgenerally to an absolute value reflecting the mass of the analytedetectable in volume of sample. However, an amount also contemplates arelative amount in comparison to another analyte amount. For example, anamount of an analyte in a sample can be an amount which is greater thana control or normal level of the analyte normally present in the sample.

The term “about” as used herein in reference to quantitativemeasurements not including the measurement of the mass of an ion, refersto the indicated value plus or minus 10%. Mass spectrometry instrumentscan vary slightly in determining the mass of a given analyte. The term“about” in the context of the mass of an ion or the mass/charge ratio ofan ion refers to +/−0.50 atomic mass unit.

The summary of the invention described above is non-limiting and otherfeatures and advantages of the invention will be apparent from thefollowing detailed description of the invention, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an exemplary chromatogram for EPA/EPA-²H₅ (internalstandard). Details are discussed in Example 4.

FIG. 2 shows an exemplary chromatogram for DHA and DHA-²H₅ (internalstandard). Details are discussed in Example 4.

FIG. 3 shows exemplary spectra demonstrating detection of EPA andEPA-²H₅ (internal standard). Details are discussed in Example 4.

FIG. 4 shows exemplary spectra demonstrating detection of DHA andDHA-²H₅ (internal standard). Details are discussed in Example 4.

FIGS. 5 and 6 show exemplary calibration curves generated for DHA andEPA, respectively, from serially diluted serum standards. Details aredescribed in Example 4.

DETAILED DESCRIPTION OF THE INVENTION

Methods are described for measuring EPA and/or DHA in a sample. Morespecifically, mass spectrometric methods are described for detecting andquantifying EPA and/or DHA in a sample. The methods may utilize APCI toionize underivatized EPA and/or DHA in the sample prior to detection bymass spectrometry.

The methods may use an on-line analytical liquid chromatographytechnique, such as high performance liquid chromatography (HPLC), toperform a purification of DHA and/or EPA, combined with methods of massspectrometry (MS), thereby providing a high-throughput assay system fordetecting and quantifying DHA and/or EPA in a sample. Preferredembodiments are particularly well suited for application in largeclinical laboratories for automated DHA and/or EPA quantitation.

Suitable test samples for use in methods of the present inventioninclude any test sample that may contain the analyte of interest. Insome preferred embodiments, a sample is a biological sample; that is, asample obtained from any biological source, such as an animal, a cellculture, an organ culture, etc. In certain preferred embodiments,samples are obtained from a mammalian animal, such as a dog, cat, horse,etc. Particularly preferred mammalian animals are primates, mostpreferably male or female humans. Preferred samples comprise bodilyfluids such as blood, plasma, serum, saliva, cerebrospinal fluid, ortissue samples; preferably plasma (including EDTA and heparin plasma)and serum; most preferably serum. Such samples may be obtained, forexample, from a patient; that is, a living person, male or female,presenting oneself in a clinical setting for diagnosis, prognosis, ortreatment of a disease or condition.

The present invention also contemplates kits for a DHA and/or EPAquantitation assay. A kit for a DHA and/or EPA quantitation assay mayinclude a kit comprising the compositions provided herein. For example,a kit may include packaging material and measured amounts of packagedreagents, including an isotopically labeled internal standard, inamounts sufficient for at least one assay. Typically, the kits will alsoinclude instructions recorded in a tangible form (e.g., contained onpaper or an electronic medium) for using the packaged reagents for usein a DHA and/or EPA quantitation assay.

Calibration and QC pools for use in embodiments of the present inventionare preferably prepared using a matrix similar to the intended samplematrix.

Sample Preparation for Mass Spectrometric Analysis

In preparation for mass spectrometric analysis, free fatty acids(including DHA and/or EPA) in the sample may be enriched relative totheir ester counterparts by hydrolysis of fatty acid esters. Hydrolysismay be accomplished by any technique known in the art. In someembodiments, fatty acid esters in the sample are hydrolyzed bycontacting the sample with an acid, such as hydrochloric acid (HCl), andincubating at an elevated temperature, such as about 105° C. to about115° C. The incubation period may vary depending on the amount of sampleand concentration of acid used. Certain embodiments described hereinutilize an incubation period of about 90 minutes to hydrolyze 200 μL ofsample, diluted with 100 μL of internal standard, with 200 μL of 5 MHCl.

Additionally, DHA and/or EPA may be enriched relative to one or moreother components in the sample (e.g. protein) by various methods knownin the art, including for example any combination of liquidchromatography, filtration, centrifugation, thin layer chromatography(TLC), electrophoresis including capillary electrophoresis, affinityseparations including immunoaffinity separations, extraction methodsincluding ethyl acetate or methanol extraction, and the use ofchaotropic agents or any combination of the above or the like. If bothhydrolysis and purification steps are used, purification is preferablyconducted after hydrolysis.

Protein precipitation is one method of preparing a test sample,especially a biological test sample, such as serum or plasma. Proteinpurification methods are well known in the art, for example, Polson etal., Journal of Chromatography B 2003, 785:263-275, describes proteinprecipitation techniques suitable for use in methods of the presentinvention. Protein precipitation may be used to remove most of theprotein from the sample leaving vitamin D in the supernatant. Thesamples may be centrifuged to separate the liquid supernatant from theprecipitated proteins; alternatively the samples may be filtered toremove precipitated proteins. The resultant supernatant or filtrate maythen be applied directly to mass spectrometry analysis; or alternativelyto liquid chromatography and subsequent mass spectrometry analysis. Incertain embodiments, samples, such as plasma or serum, may be purifiedby a hybrid protein precipitation/liquid-liquid extraction method. Inthese embodiments, a sample is mixed with methanol, ethyl acetate, andwater, and the resulting mixture is vortexed and centrifuged. Theresulting supernatant is removed, dried to completion and reconstitutedin a suitable solvent. In certain embodiments described herein, thesolvent used to reconstitute the dried supernatant is ethanol.

Another method of sample purification that may be used prior to massspectrometry is liquid chromatography (LC). Certain methods of liquidchromatography, including HPLC, rely on relatively slow, laminar flowtechnology. Traditional HPLC analysis relies on column packing in whichlaminar flow of the sample through the column is the basis forseparation of the analyte of interest from the sample. The skilledartisan will understand that separation in such columns is a diffusionalprocess and may select LC, including HPLC, instruments and columns thatare suitable for use with DHA and/or EPA. The chromatographic columntypically includes a medium (i.e., a packing material) to facilitateseparation of chemical moieties (i.e., fractionation). The medium mayinclude minute particles, or may include a monolithic material withporous channels. A surface of the medium typically includes a bondedsurface that interacts with the various chemical moieties to facilitateseparation of the chemical moieties. One suitable bonded surface is ahydrophobic bonded surface such as an alkyl bonded, cyano bondedsurface, or highly pure silica surface. Alkyl bonded surfaces mayinclude C-4, C-8, C-12, or C-18 bonded alkyl groups. In preferredembodiments, the column is a C-18 alkyl bonded column (such as a BDSHypersil C18 column from Thermo Scientific). The chromatographic columnincludes an inlet port for receiving a sample and an outlet port fordischarging an effluent that includes the fractionated sample. Thesample may be supplied to the inlet port directly, or from an extractioncolumn, such as an on-line SPE cartridge or a TFLC extraction column.

In one embodiment, the sample may be applied to the LC column at theinlet port, eluted with a solvent or solvent mixture, and discharged atthe outlet port. Different solvent modes may be selected for eluting theanalyte(s) of interest. For example, liquid chromatography may beperformed using a gradient mode, an isocratic mode, or a polytyptic(i.e. mixed) mode. During chromatography, the separation of materials iseffected by variables such as choice of eluent (also known as a “mobilephase”), elution mode, gradient conditions, temperature, etc.

In certain embodiments, an analyte may be purified by applying a sampleto a column under conditions where the analyte of interest is reversiblyretained by the column packing material, while one or more othermaterials are not retained. In these embodiments, a first mobile phasecondition may be employed where the analyte of interest is retained bythe column, and a second mobile phase condition may subsequently beemployed to remove retained material from the column, once thenon-retained materials are washed through. Alternatively, an analyte maybe purified by applying a sample to a column under mobile phaseconditions where the analyte of interest elutes at a differential ratein comparison to one or more other materials. Such procedures may enrichthe amount of one or more analytes of interest relative to one or moreother components of the sample.

In one preferred embodiment, HPLC is conducted with an alkyl bondedanalytical column chromatographic system. In certain preferredembodiments, a C-18 alkyl bonded column (such as a BDS Hypersil C18column from Thermo Scientific) is used. In certain embodiments, HPLC isperformed using 20 mM ammonium acetate as mobile phase A and 100%acetonitrile as mobile phase B.

By careful selection of valves and connector plumbing, two or morechromatography columns may be connected as needed such that material ispassed from one to the next without the need for any manual steps. Inpreferred embodiments, the selection of valves and plumbing iscontrolled by a computer pre-programmed to perform the necessary steps.Most preferably, the chromatography system is also connected in such anon-line fashion to the detector system, e.g., an MS system. Thus, anoperator may place a tray of samples in an autosampler, and theremaining operations are performed under computer control, resulting inpurification and analysis of all samples selected.

In some embodiments, an extraction column may be used for purificationof DHA and/or EPA prior to mass spectrometry. In such embodiments,samples may be extracted using an extraction column which captures theanalyte, then eluted and chromatographed on a second extraction columnor on an analytical HPLC column prior to ionization. For example, sampleextraction with a TFLC extraction column may be accomplished with alarge particle size (50 μm) packed column. Sample eluted off of thiscolumn may then be transferred to an HPLC analytical column for furtherpurification prior to mass spectrometry. Because the steps involved inthese chromatography procedures may be linked in an automated fashion,the requirement for operator involvement during the purification of theanalyte can be minimized. This feature may result in savings of time andcosts, and eliminate the opportunity for operator error.

In some embodiments, purification of DHA and/or EPA is accomplished withliquid-liquid extraction. Liquid/liquid extraction may be accomplishedby adding a suitable quantity of an organic solvent, such as 10% ethylacetate in hexane, to the sample. This mixture is then vortexed andchilled, and the organic layer is decanted off for further analysis. Insome embodiments, DHA and/or EPA in the sample may be purified byliquid/liquid extraction followed by liquid chromatography prior to massspectrometric analysis.

Detection and Quantitation by Mass Spectrometry

Mass spectrometry is performed using a mass spectrometer, which includesan ionization source for ionizing the fractionated sample and creatingcharged molecules for further analysis. For example, ionization of thesample may be performed by electron ionization, chemical ionization,electrospray ionization (ESI), photon ionization, atmospheric pressurechemical ionization (APCI), photoionization, atmospheric pressurephotoionization (APPI), laser diode thermal desorption (LDTD), fast atombombardment (FAB), liquid secondary ionization (LSI), matrix assistedlaser desorption ionization (MALDI), field ionization, field desorption,thermospray/plasmaspray ionization, surface enhanced laser desorptionionization (SELDI), inductively coupled plasma (ICP) and particle beamionization. In preferred embodiments, DHA and/or EPA in the sample areionized by APCI.

Mass spectrometric techniques may be conducted in positive or negativeionization mode. In preferred embodiments, DHA and/or EPA are ionized byAPCI in negative ionization mode.

In mass spectrometry techniques generally, after the sample has beenionized, the positively or negatively charged ions created thereby maybe analyzed to determine a mass-to-charge ratio. Suitable analyzers fordetermining mass-to-charge ratios include quadrupole analyzers, iontraps analyzers, and time-of-flight analyzers. Exemplary ion trapmethods are described in Bartolucci, et al., Rapid Commun. MassSpectrom. 2000, 14:967-73.

Ions in a MS system may be detected using several detection modes. Forexample, selected ions may be detected, i.e. using a selective ionmonitoring mode (SIM), or alternatively, mass transitions resulting fromcollision induced dissociation or neutral loss may be monitored, e.g.,multiple reaction monitoring (MRM) or selected reaction monitoring(SRM). Preferably, the mass-to-charge ratio is determined using aquadrupole analyzer. For example, in a “quadrupole” or “quadrupole iontrap” instrument, ions in an oscillating radio frequency fieldexperience a force proportional to the DC potential applied betweenelectrodes, the amplitude of the RF signal, and the mass/charge ratio.The voltage and amplitude may be selected so that only ions having aparticular mass/charge ratio travel the length of the quadrupole, whileall other ions are deflected. Thus, quadrupole instruments may act asboth a “mass filter” and as a “mass detector” for the ions injected intothe instrument.

One may enhance the resolution of the MS technique by employing “tandemmass spectrometry,” or “MS/MS”. In this technique, a precursor ion (alsocalled a parent ion) generated from a molecule of interest can befiltered in an MS instrument, and the precursor ion subsequentlyfragmented to yield one or more fragment ions (also called daughter ionsor product ions) that are then analyzed in a second MS procedure. Bycareful selection of precursor ions, only ions produced by certainanalytes are passed to the fragmentation chamber, where collisions withatoms of an inert gas produce the fragment ions. Because both theprecursor and fragment ions are produced in a reproducible fashion undera given set of ionization/fragmentation conditions, the MS/MS techniquemay provide an extremely powerful analytical tool. For example, thecombination of filtration/fragmentation may be used to eliminateinterfering substances, and may be particularly useful in complexsamples, such as biological samples.

Alternate modes of operating a tandem mass spectrometric instrumentinclude product ion scanning and precursor ion scanning. For adescription of these modes of operation, see, e.g., E. Michael Thurman,et al., Chromatographic-Mass Spectrometric Food Analysis for TraceDetermination of Pesticide Residues, Chapter 8 (Amadeo R.Fernandez-Alba, ed., Elsevier 2005) (387).

The results of an analyte assay may be related to the amount of theanalyte in the original sample by numerous methods known in the art. Forexample, given that sampling and analysis parameters are carefullycontrolled, the relative abundance of a given ion may be compared to atable that converts that relative abundance to an absolute amount of theoriginal molecule. Alternatively, external standards may be run with thesamples, and a standard curve constructed based on ions generated fromthose standards. Using such a standard curve, the relative abundance ofa given ion may be converted into an absolute amount of the originalmolecule. In certain preferred embodiments, an internal standard is usedto generate a standard curve for calculating the quantity of DHA and/orEPA in the sample. Methods of generating and using such standard curvesare well known in the art and one of ordinary skill is capable ofselecting an appropriate internal standard. For example, in someembodiments one or more isotopically labeled DHA and/or EPA (e.g.,DHA-²H₅ and EPA-²H₅) may be used as internal standards. Numerous othermethods for relating the amount of an ion to the amount of the originalmolecule will be well known to those of ordinary skill in the art.

One or more steps of the methods may be performed using automatedmachines. In certain embodiments, one or more purification steps areperformed on-line, and more preferably all of the purification and massspectrometry steps may be performed in an on-line fashion.

In certain embodiments, such as MS/MS, where precursor ions are isolatedfor further fragmentation, collision activated dissociation (CAD) isoften used to generate fragment ions for further detection. In CAD,precursor ions gain energy through collisions with an inert gas, andsubsequently fragment by a process referred to as “unimoleculardecomposition.” Sufficient energy must be deposited in the precursor ionso that certain bonds within the ion can be broken due to increasedvibrational energy.

In some preferred embodiments, DHA and/or EPA in a sample are detectedand/or quantitated using MS as follows. The samples are first purifiedby liquid-liquid extraction. Then, the purified sample is subjected toliquid chromatography, preferably on an analytical column (such as aHPLC column) and the flow of eluted DHA and/or EPA from thechromatographic column is directed to the ionization source of an MSanalyzer. DHA and/or EPA from the chromatographic column are ionized viaAPCI in negative ionization mode. The generated ions pass through theorifice of the instrument and enter a quadrupole. The quadrupole acts asa mass filter, allowing selection of ions (i.e., selection of“precursor” and “fragment” ions in Q1 and Q3, respectively) based ontheir mass to charge ratio (m/z). The quadrupole selects for ions withthe mass to charge ratios of DHA and/or EPA ions of interest. Ions withthe correct mass/charge ratios are allowed to pass the quadrupole andcollide with the detector.

As ions collide with the detector they produce a pulse of electrons thatare converted to a digital signal. The acquired data is relayed to acomputer, which plots counts of the ions collected versus time. Theareas under the peaks corresponding to particular ions, or the amplitudeof such peaks, may be measured and correlated to the amount of theanalyte of interest. In certain embodiments, the area under the curves,or amplitude of the peaks, for DHA and/or EPA ions are measured todetermine the amount of DHA and/or EPA in the original sample. Asdescribed above, the relative abundance of a given ion may be convertedinto an absolute amount of the original analyte using calibrationstandard curves based on peaks of one or more ions of an internalmolecular standard.

The following Examples serve to illustrate the invention. These Examplesare in no way intended to limit the scope of the methods. In particular,the Examples demonstrate quantitation of DHA and/or EPA by massspectrometry, and the use of DHA-²H₅ and EPA-²H₅ as internal standards.The use of DHA-²H₅ and EPA-²H₅ as internal standards are not meant to belimiting in any way. Any appropriate chemical species, easily determinedby one in the art, may be used as an internal standard.

EXAMPLES Example 1 Reagent Preparation

Three control solutions (at low, medium, and high DHA and EPAconcentrations) were prepared for use in the following Examples. Thecontrol solutions were prepared by spiking DHA and EPA into Biocel serumsamples to achieve final concentrations of 45 μmol/L DHA and 25 μmol/LEPA for the low level control, 120 μmol/L DHA and 65 μmol/L EPA for themid level control, and 400 μmol/L DHA and 125 μmol/L EPA for the highlevel control.

A standard stock solution was similarly prepared by spiking DHA and EPAinto Biocel serum samples to target concentrations of 600 μmol/L DHA and300 μmol/L EPA. A series of 1:2 dilutions of the standard stock solutionwas prepared for use below in generation of a calibration curve(discussed in Example 4).

An internal standard solution was prepared by diluting both DHA-²H₅ andEPA-²H₅ in the same ethyl alcohol solution to concentrations of about150 μmol/L and 50 μmol/L, respectfully.

Example 2 Hydrolysis of Fatty Acid Esters and Liquid-Liquid Extraction

The following hydrolysis and liquid-liquid extraction techniques wereconducted on controls, standards, and patient serum samples to preparesamples for mass spectrometric analysis. Plasma samples were also testedwith similar results (not shown).

First, 100 μL of the DHA-²H₅ and EPA-²H₅ internal standard mixturedescribed above was mixed with 200 μIL aliquots of each standard,control, and patient sample. 200 μL of 5 M HCL was added to eachmixture, and the resulting mixtures vortexed for about 10 seconds. Theacidified mixtures were then placed in an oven and incubated at about105° C.-115° C. for about 90 minutes.

After incubation was complete, the mixtures were cooled in a 2° C.-8° C.refrigerator for 5-10 minutes. 3.5 mL of 10% ethyl acetate in hexane wasadded to each of the cooled samples; the resulting mixtures vortexed for4 minutes and centrifuged at about 3000 rpm for about 5 minutes. Aftercentrifugation, the samples were placed in a methanol/dry ice bath forabout 5 minutes to freeze the aqueous later. The organic layer was thendecanted off, dried to completion under a flowing nitrogen gas manifold,and reconstituted in 150 μL of ethanol.

The resulting samples were transferred to HPLC vials and placed in anautosampler for analysis.

Example 3 Purification of DHA and EPA with Liquid Chromatography

Sample injection was performed with an Agilent Technologies G1367BAutosampler.

The autosampler system automatically injected an aliquot of the aboveprepared reconstituted samples into a Thermo Scientific BDS Hypersil C18HPLC column (3 μm particle size, 100×2.1 mm, from Thermo Scientific). AnHPLC gradient was applied to the analytical column, to separate DHA andEPA from other components in the sample. Mobile phase A was 20 mMammonium acetate and mobile phase B was 100% acetonitrile. The HPLCgradient started with a 54% solvent A which was ramped to 82% inapproximately 2.5 minutes, and held for approximately 30 seconds, beforebeing ramped back down to 54% over the next 30 seconds. Column flow rateduring solvent application was about 0.75 mL/min. DHA and EPA wereobserved to elute off the column at approximately 1.40 minutes into thegradient profile.

Example 4 Detection and Quantitation of DHA and EPA by MS

MS was performed on the above eluted samples using an Agilent 6130Single Quadrupole Mass Spectrometer. Liquid solvent/analyte exiting theanalytical column flowed to the ionization interface of the MS/MSanalyzer. The solvent/analyte mixture was converted to vapor in thetubing of the interface. Analytes in the nebulized solvent were ionizedby APCI.

Ions passed to the quadrupole mass selector (Q1), which selected DHA andEPA ions with mass-to-charge ratios of 327.2±0.5 m/z and 301.2±0.5 m/z,respectively. The selected DHA and EPA ions then traveled to a detectorfor counting. Mass spectrometer settings used for this Example are shownin Table 1. Simultaneously, the same process using isotope dilution massspectrometry was carried out with internal standards, DHA-²H₅ andEPA-²H₅. The masses monitored for detection and quantitation duringvalidation on negative polarity are shown in Table 2.

TABLE 1 Mass Spectrometer Settings for Detection of DHA, EPA, DHA-²H₅(internal standard), and EPA-²H₅ (internal standard) (NegativeIonization) Mass Spectrometric Instrument Settings Gas Temperature 345°C. Vaporizer Temperature 245° C. Drying Gas Flow 10.0 L/min NebulizerPressure 50 psig Vcap (positive) 4000 V Vcap (negative) 2400 V Vcharge(positive) 2000 V Vcharge (negative) 1000 V Corona (positive) 5.0 μACorona (negative) 40 μA

TABLE 2 Mass-to-Charge ratios monitored for DHA, EPA, DHA-²H₅ (internalstandard), and EPA-²H₅ (internal standard) (Negative Ionization) AnalyteIon (m/z) DHA 327.2 ± 0.5 EPA 301.2 ± 0.5 DHA-²H₅ 332.1 ± 0.5 EPA-²H₅306.2 ± 0.5

Exemplary chromatograms for EPA/EPA-²H₅ (internal standard) andDHA/DHA-²H₅ (internal standard) are shown in FIGS. 1 and 2,respectively.

Exemplary spectra from the mass spectrometric analysis of EPA/EPA-²H₅(internal standard) and DHA/DHA-²H₅ (internal standard) generated asdescribed above are shown in FIGS. 3 and 4, respectively. The spectrawere collected by scanning Q1 across a m/z range of about 300 to 333 forEPA, and about 321 to 333 for DHA.

Calibration curves were prepared for the quantitation of DHA and EPA inserum by analysis of standards at about 600 μmol/L DHA and 300 μmol/LEPA (std. 1), 300 μmol/L DHA and 150 μmol/L EPA (std. 2), 150 μmol/L DHAand 75 μmol/L EPA (std. 3), 75 μmol/L DHA and 37.5 μmol/L EPA (std. 4),37.5 μmol/L DHA and 18.8 μmol/L EPA (std. 5), 18.8 μmol/L DHA and 9.4μmol/L EPA (std. 6), and 9.4 μmol/L DHA and 4.7 μmol/L EPA (std. 7).Exemplary calibration curves for the determination of EPA and DHA inserum specimens are shown in FIGS. 5 and 6, respectively. Analysis ofthe data generated for these standards demonstrates that the assayexhibits linear response for DHA in the concentration range of about0.912 μmol/L to 600 μmol/L (correlation of about 0.99992), and for EPAin the concentration range of about 2.278 μmol/L to 300 μmol/L(correlation of about 0.99977).

Example 5 Analytical Precision for MS Determination of DHA and EPA

Intra-assay precision studies for DHA were conducted by analyzingmultiple aliquots of the three control solutions described in Example 1according to the analytical techniques described in Examples 2-4.Statistical analysis of the results of these studies gave CVs of 4.9%,3.6%, and 2.9%, for the low, mid, and high concentration levels,respectively. For all three controls, the observed standard deviation(n=24 samples) was less than one quarter of the total allowable error.Additionally, pooled within-run CVs from the inter-assay precision study(n=25 samples) were 3.6%, 3.2%, and 2.6%, respectively. Again, allobserved within-run standard deviations were less than one quarter ofthe total allowable error.

Intra-assay precision studies for EPA were conducted by analyzingmultiple aliquots of the three control solutions described in Example 1according to the analytical techniques described in Examples 2-4.Statistical analysis of the results of these studies gave CVs of 3.0%,1.1%, and 2.0%, for the low, mid, and high concentration levels,respectively. For all three controls, the observed standard deviation(n=24 samples) was less than one quarter of the total allowable error.Additionally, pooled within-run CVs from the inter-assay precision study(n=25 samples) were 2.9%, 3.5%, and 2.6%, respectively. Again, allobserved within-run standard deviations were less than one quarter ofthe total allowable error.

Inter-assay precision studies for DHA were conducted by analyzingmultiple aliquots of the three control solutions described in Example 1according to the analytical techniques described in Examples 2-4.Statistical analysis of the results of these studies gave CVs of 5.7%,4.8%, and 3.7%, for the low, mid, and high concentration levels,respectively. For all three controls, the observed standard deviation(n=24 samples) was less than one half of the total allowable error.Additionally, pooled within-run CVs from the inter-assay precision study(n=25 samples) were 3.92%, 4.64%, and 6.06%, respectively.

Inter-assay precision studies for EPA were conducted by analyzingmultiple aliquots of the three control solutions described in Example 1according to the analytical techniques described in Examples 2-4.Statistical analysis of the results of these studies gave CVs of 4.49%,4.14%, and 4.44%, for the low, mid, and high concentration levels,respectively. For all three controls, the observed standard deviation(n=24 samples) was less than one half of the total allowable error.Additionally, pooled within-run CVs from the inter-assay precision study(n=25 samples) were 4.90%, 5.31%, and 4.96%, respectively.

Example 6 Analytical Sensitivity: Limit of Blank (LOB), Lower Limit ofQuantitation (LLOQ), and Limit of Detection (LOD)

The limit of blank (LOB) is defined as the mean value of analysis ofseveral blank samples plus two standard deviations. The LOB wasdetermined for DHA to be 0.236 μmol/L, and for EPA to be 0.654 μmol/L.Data generated for the determination of LOB of DHA and EPA are shown inTable 4.

The lower limit of quantitation (LLOQ) is the point where measurementsbecome quantitatively meaningful. The analyte response at this LLOQ isidentifiable, discrete and reproducible with a standard deviation ofless than TEa/3. The LLOQ was determined by analyzing the low, medium,and high concentration controls prepared in Example 1. Five replicatesof each control were analyzed five times. The LLOQ was determined forDHA to be 0.912 μmol/L, and for EPA to be 2.278 μmol/L. Data generatedfor the determination of LLOQ of DHA and EPA are shown in Table 3.

TABLE 3 Determination of Lower Limit of Quantitation of DHA and EPA DHAEPA Controls Controls (μmol/L) (μmol/L) Run Low Medium High Run LowMedium High 1 0.972 2.193 4.334 1 0.534 1.193 2.329 0.933 2.192 4.4550.488 1.207 2.204 0.907 2.202 4.225 0.616 1.167 2.255 0.942 2.175 4.3100.558 1.169 2.301 0.941 2.173 4.241 0.504 1.134 2.252 2 0.935 2.0754.174 2 0.555 1.175 2.168 0.920 2.072 4.068 0.595 1.155 2.223 0.9372.044 4.185 0.506 1.142 2.283 0.917 2.070 4.480 0.552 1.110 2.261 0.9081.976 4.145 0.515 1.123 2.250 3 0.943 2.430 4.640 3 0.579 1.347 2.4980.883 2.399 4.571 0.403 1.384 2.440 0.982 2.375 4.537 0.530 1.370 2.5180.923 2.376 4.565 0.533 1.321 2.476 0.956 2.505 4.665 0.398 1.407 2.5564 0.905 1.964 4.572 4 0.546 0.979 2.199 0.853 1.978 4.571 0.370 1.0062.272 0.797 1.992 4.390 0.279 0.944 2.246 0.877 1.977 4.445 0.274 0.9922.121 0.893 1.939 4.530 0.226 0.969 2.250 5 0.923 2.101 4.442 5 0.4661.005 2.103 0.878 2.094 4.414 0.484 0.996 2.123 0.885 2.086 4.689 0.3880.991 2.123 0.888 2.062 4.449 0.394 1.072 2.267 0.902 2.129 4.471 0.4120.975 2.232 Count 25 25 25 Count 25 25 25 Mean 0.912 2.143 4.423 Mean0.468 1.133 2.278 SD 0.039 0.160 0.172 SD 0.105 0.144 0.127 TEa/3 0.0680.161 0.332 TEa/3 0.034 0.083 0.167 LOB 0.236 LOB 0.654 LOQ 0.912 LOQ2.278

The limit of detection (LOD) is the point where a measured value islarger than the uncertainty associated with it and is definedarbitrarily as four standard deviations (SD) from the means signal fromzero concentration. A blank was run in 20 replicates and the resultingarea ratios were statistically analyzed. From this analysis, the LOD forDHA and EPA were determined to be about 0.258 μmol/L and 0.67 μmol/L,respectively. Data collected to determine LOD for each analyte is shownin Table 6.

TABLE 4 Determination of Limit of Detection of DHA and EPA DHA (ResponseRatio) EPA (Response Ratio) Replicate # (μmol/L) (μmol/L)  1 0.205 0.633 2 0.228 0.636  3 0.221 0.657  4 0.238 0.632  5 0.193 0.630  6 0.2050.634  7 0.210 0.643  8 0.215 0.633  9 0.222 0.633 10 0.218 0.640 110.220 0.637 12 0.208 0.654 13 0.197 0.635 14 0.224 0.629 15 0.215 0.63916 0.207 0.628 17 0.205 0.626 18 0.209 0.649 19 0.228 0.638 20 0.2100.633 Mean 0.214 0.637 SD 0.011 0.008 LOB 0.236 μmol/L 0.654 μmol/L LOD0.258 μmol/L 0.670 μmol/L

Example 7 Analyte Measurement Range (AMR)

A sample with a low DHA value (mean 60.42 μmol/L) and a sample with ahigh DHA value (mean 600.14 μmol/L) were mixed in various proportionsand analyzed according to the method outlined in the Examples above. Theobserved results were compared to the expected (calculated) results. Forall admixtures, the difference between the observed mean DHA value andthe expected (calculated) value was less than TEa/4. Results of thesestudies are presented in Table 5.

TABLE 5 Comparison of Observed and Expected Values for Various Levels ofDHA in Mixed Serum Samples Mean Expected Sample Mix Observed(Calculated) (% High/% Low) (μmol/L) (μmol/L) TEa/4 Difference  0/100 60.42  60.42 3.4   0.00 25/75 188.66 195.35 10.61 −6.75 50/50 336.46330.28 18.93   6.18 75/25 456.35 465.21 25.67 −8.86 100/0  600.14 600.1433.76   0.00

A sample with a low EPA value (mean 10.92 μmol/L) and a sample with ahigh EPA value (mean 282.52 μmol/L) were mixed in various proportionsand analyzed according to the method outlined in the Examples above. Theobserved results were compared to the (calculated) expected results. Forall admixtures, the difference between the observed mean EPA value andthe expected (calculated) value was less than TEa/4. Results of thesestudies are presented in Table 6.

TABLE 6 Comparison of Observed and Expected Values for Various Levels ofEPA in Mixed Serum Samples Mean Expected Sample Mix Observed(Calculated) (% High/% Low) (μmol/L) (μmol/L) TEa/4 Difference  0/100 10.92  10.92  0.60 0.00 25/75  81.02  78.82  4.46 2.19 50/50 151.73146.72  8.34 5.00 75/25 218.75 214.62 12.03 4.13 100/0  282.52 282.5215.54 0.00

Example 8 Recovery Studies for DHA and EPA

Six serum samples with low baseline DHA and EPA levels were selected foranalysis according to the method described in the above Examples forrecovery studies. A known amount of standard (comprising known amountsof DHA and EPA) was added to each sample to establish a target level toassess recoveries. Each spiked sample was tested in quadruplicate. Thedifference between the mean of the four measurements and its targetvalue were calculated. The results for DHA and EPA are summarized inTables 7-8, respectively:

TABLE 7 Recovery of DHA in Serum Samples Target Value Mean ObservedSample (μmol/L) (μmol/L) TEa/4 Difference 1 155.54 141.14  8.75 14.4  2167.57 155.79  9.43 11.78 3 233.74 221.17 13.15 12.57 4 343.59 333.4619.33 10.13 5  72.50  62.68  4.08  9.82 6 586.33 585.41 32.98  0.92

For analysis of DHA, the difference between the target amount and theobserved amount was less than TEa/4 for samples 3, 4, and 6. However,the difference between the observed and targeted values for all sampleswas not large enough to be clinically significantly.

TABLE 8 Recovery of EPA in Serum Samples Target Value Mean ObservedSample (μmol/L) (μmol/L) TEa/4 Difference 1  35.84  33.38  1.97 2.46 2 48.51  47.23  2.67 1.28 3 124.02 125.09  6.82 1.07 4 249.98 253.2613.75 3.28 5  11.63  10.26  0.64 1.37 6 527.08 530.96 28.99 3.88

For analysis of EPA, the difference between the target amount and theobserved amount was less than TEa/4 for samples 3, 4, and 6. However,the difference between the observed and targeted values for all sampleswas not large enough to be clinically significantly.

Example 9 Interference Studies

Hemolysis Interference: The effects of hemolysis in the assay wereevaluated by spiking various levels of washed red cells into the low andhigh DHA and EPA controls (described in Example 1) to mimic variousdegrees of hemolysis (low red, red, cherry red, dark red). All sampleswere analyzed in quadruplet for DHA and EPA. The results of theseanalyses are shown in Tables 9 and 10.

TABLE 9 Hemolysis Interference Studies for DHA DHA Low Control MeanBaseline Mean Observed Sample (μmol/L) (μmol/L) TEa/4 Difference Low Red40.26 42.26 2.26  2.00 Red 40.26 42.67 2.26  2.41 Cherry Red 40.26 46.782.26  6.52 Dark Red 40.26 52.22 2.26 11.96 DHA High Control MeanBaseline Mean Observed Sample (μmol/L) (μmol/L) TEa/4 Difference Low Red347.76 346.96 19.56  0.80 Red 347.76 339.01 19.56  8.75 Cherry Red347.76 353.76 19.56  6.00 Dark Red 347.76 360.40 19.56 12.64

TABLE 10 Hemolysis Interference Studies for EPA EPA Low Control MeanBaseline Mean Observed Sample (μmol/L) (μmol/L) TEa/4 Difference Low Red23.64 24.35 1.30 0.71 Red 23.64 24.68 1.30 1.04 Cherry Red 23.64 23.701.30 0.06 Dark Red 23.64 26.02 1.30 2.38 EPA High Control Mean BaselineMean Observed Sample (μmol/L) (μmol/L) TEa/4 Difference Low Red 108.66108.74 5.98 0.08 Red 108.66 109.98 5.98 1.32 Cherry Red 108.66 111.335.98 2.67 Dark Red 108.66 109.33 5.98 0.67

Although the high control data suggest that hemolysis has little or noeffect on samples where the levels of DHA and EPA are high, therelatively large effect seen in samples with low DHA and EPA valuesprevents acceptability of hemolyzed samples.

Bilirubin Interference: The effects of bilirubin in the assay wereevaluated by spiking various levels of bilirubin into the low level DHAand EPA control (described in Example 1) to mimic mildly (5 mg/dLbilirubin), moderately (10 mg/dL bilirubin), and grossly (40 mg/dLbilirubin) icteric samples. All samples were analyzed in quadruplet forDHA and EPA. The results of these analyses are shown in Tables 11 and12.

TABLE 11 Bilirubin Interference Studies for DHA DHA Low Control MeanBaseline Mean Observed Sample (μmol/L) (μmol/L) TEa/4 Difference Mild82.10 84.94 4.61 2.93 Moderate 82.10 85.36 4.61 3.35 Gross 82.10 85.624.61 3.61

TABLE 12 Bilirubin Interference Studies for EPA EPA Low Control MeanBaseline Mean Observed Sample (μmol/L) (μmol/L) TEa/4 Difference Mild49.31 52.32 2.71 3.01 Moderate 49.31 51.91 2.71 2.60 Gross 49.31 51.482.71 2.17

For both DHA and EPA, the differences between the observed mean valuesand the expected value were greater than TEa/4 at all levels of icterus,except for EPA in the midly icteric sample. Thus, icteric samples areunacceptable for this assay.

Example 10 Development of Reference Intervals for DHA and EPA

Specimens were collected from 120 sources (60 female and 60 male) forDHA and 119 sources (60 female and 59 male) for EPA from apparentlyhealthy, ambulatory, community dwelling, and non-medicated adults.Exclusion criteria for development of the reference range populationwere: no fish oil, DHA, or EPA dietary supplements, or patients thatreport a diet rich in seafood (i.e., greater than 1 serving per week).

The above specimens were analyzed for DHA and EPA according to theprocedures outlined in Examples 1-3. The resulting data were neither agenor gender dependent, and were Gaussian, with logarithmictransformation. Reference ranges were defined as the logarithmic(transformed) mean±2 standard deviations. The reference range for DHA isabout 48.98-250.03 μmol/L. The reference range for EPA is about6.43-87.90 μmol/L.

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

The methods illustratively described herein may suitably be practiced inthe absence of any element or elements, limitation or limitations, notspecifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof. It is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the invention embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the methods. This includes the genericdescription of the methods with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the methods are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

That which is claimed is:
 1. A method for determining an amount ofdocosahexaenoic acid (DHA) and/or eicosapentaenoic acid (EPA) in asample by mass spectrometry, the method comprising: (i) hydrolyzing thesample containing DHA and/or EPA with an acid; (ii) extracting DHAand/or EPA from the sample by liquid-liquid extraction; (iii) ionizingDHA and/or EPA from the sample to generate one or more ions detectableby mass spectrometry; (iv) determining an amount of DHA and/or EPA ionsby single mass spectrometry; and (v) relating the amount of DHA and/orEPA ions to the amount of DHA and/or EPA in the sample, wherein a limitof detection of the method for DHA is between about 1 μmol/L and 0.25μmol/L, and a limit of detection of the method for EPA is between about1 μmol/L and 0.67 μmol/L.
 2. The method of claim 1, wherein saidionizing comprises atmospheric pressure chemical ionization (APCI). 3.The method of claim 2, wherein said APCI is in negative ionization mode.4. The method of claim 1, wherein said ionizing comprises electrosprayionization (ESI).
 5. The method of claim 1, wherein said one or moreions comprise an ion with a mass to charge ratio (m/z) of 327.2±0.5. 6.The method of claim 1, wherein said one or more ions comprise an ionwith a mass to charge ratio (m/z) of 301.2±0.5.
 7. The method of claim1, wherein said sample comprises human serum or plasma.
 8. The method ofclaim 1, wherein said acid is hydrochloric acid.
 9. The method of claim1, wherein said DHA and/or EPA is purified by liquid chromatographyprior to ionization.
 10. The method of claim 9, wherein said liquidchromatography comprises high performance liquid chromatography (HPLC).11. The method of claim 1, wherein said method comprises determining anamount of an internal standard.
 12. The method of claim 11, wherein saidinternal standard is a deuterated omega-3 fatty acid.
 13. The method ofclaim 12, wherein said deuterated omega-3 fatty acid is DHA-²H₅.
 14. Themethod of claim 12, wherein said deuterated omega-3 fatty acid isEPA-²H₅.